Inhibition of chemically induced erythrodifferentiation in friends erythroleukemia cells by d,1-Propranolol

Dimethyl sulfoxide (2%), hexamethylene bisacetamide (4mM) and butyric acid (2mM) were potent inducers of erythrodifferentiation in Friend erythroleukemia cell lines, 5–18 and C19TK. Hydrocortisone (1μM) markedly inhibited dimethyl sulfoxide induced hemoglobin production in both 5–18 and C19TK cells. d,1-Propranolol (25–50μM) markedly inhibited both dimethyl sulfoxide and hexamethylene bisacetamide induced erythrodifferentiation in 5–18 cells but not in C19TK cells. Addition of either hydrocortisone or propranolol as late as 48 hrs after dimethyl sulfoxide addition still resulted in significant inhibition of hemoglobin synthesis in 5–18 cells. Although the mechanism of action of propranolol is not known, modulation of the β adrenergic receptor is apparently not involved since practolol failed to inhibit either dimethyl sulfoxide or hexamethylene bisacetamide induced erythrodifferentiation in 5–18 cells nor did isoproternol induce hemoglobin synthesis.     

Biochemical changes after hepatic injury by allyl alcohol and N-hydroxy-2-acetylaminofluorene.

Administration of hepatotoxic doses of allyl alcohol and N-hydroxy-2-acetylaminofluorene (N-OH-AAF) TO adult male rats produced periportal necrosis and functional derangement of the hepatic endoplasmic reticulum within 24 h. The rates of N-demethylation of ethylmorphine and p-hydroxylation of aniline were decreased 6 h following allyl alcohol administration, but cytochromes P-450 and b5 were unchanged. In contrast, administration of NOH-AAF decreased cytochromes P-450 and b5 and the rate of aniline p-hydroxylation, but did not change the rate of N-demethylation of ethylmorphine or the activities of cytochrome c reductase and glucose-6-phosphatase. No decrease was observed in the activity of the cytosol enzyme, DT diaphorase, following allyl alcohol treatment. The changes by these periportal hepatotoxins were compared with those produced both by central and midzonal hepatotoxins and with changes occurring in the liver after surgical partial hepatectomy.     

Evidence for a common cell of origin for primitive epithelial cells isolated from rat liver and pancreas.

The appearance of differentiated hepatocytes in the adult rat pancreas as well as pancreatic-type tissue in the adult rat liver can be experimentally induced (Reddy et al.: J. Cell Biol., 98:2082-2090, 1984; Rao et al., J. Histochem. Cytochem., 34:197-201, 1986). These observations suggest a lineage relationship between cell compartments present in rat liver and pancreas. The present data demonstrate that epithelial cell lines with almost identical phenotypes can be established from adult rat liver and pancreas. The established cell lines showed similar morphologies as established by light- and electron-microscopic studies. The cell lines showed a unique expression pattern of intermediate filament proteins. Vimentin, actin, and beta-tubulin were present in all cell lines. In addition, simple epithelial type II cytokeratins 7 and 8 were found to be coexpressed with the type I cytokeratin 14 in several of the cell lines. Neither the type I cytokeratins 18 and 19, which are the normal partners for cytokeratins 8 and 7 in filament formation, nor the type II cytokeratin 5 could be detected despite the fact that filaments were formed by both cytokeratins 8 and 14. This suggests that cytokeratin 14 acts as an indiscriminate type I cytokeratin in filament formation in the established cell lines. The cell lines expressed the same sets of LDH and aldolase isoenzymes and identical sets of glutathione transferase subunits. In addition, the epithelial cell lines from liver and pancreas were equally sensitive to the growth-inhibitory effects of TGF-beta 1. No expression of tissue- or cell-specific proteins such as alpha-fetoprotein, albumin, amylase, elastase, or gamma-glutamyl transpeptidase were detected. The almost identical phenotypes of the hepatic and pancreatic cell lines suggest that they may be derived from a common primitive epithelial cell type present in both rat liver and pancreas. In contrast to parenchymal cells, these cells have an extended capacity for proliferation in vitro and may represent a progeny from a "precursor" or "stem" cell compartment in vivo.     

Expression of microsomal and cytosolic epoxide hydrolases in cultured rat hepatocytes and hepatoma cell lines

Microsomal epoxide hydrolase activity, determined using benzpyrene 4,5-oxide and styrene 7,8-oxide, increased in cultured hepatocytes compared to freshly isolated cells. In contrast, cytosolic epoxide hydrolase activity, assayed using trans-stilbene oxide, had decreased 80% by 24 hr and was barely detectable after 96 hr in culture. There was no difference in enzyme activity between freshly isolated hepatocytes and the two rat hepatoma cell lines McA-RH 7777 and H4-II-E, when styrene 7,8-oxide was used as substrate. However, benzpyrene 4,5-oxide hydrolase activity of the McA-RH 7777 and H4-II-E cell lines were 55 and 10%, respectively, of freshly isolated hepatocytes. These results show that hepatoma cell lines provide a suitable system for studying the regulation of both the microsomal and cytosolic epoxide hydrolase enzymes.     

Inter- and intraclonal variability of polypeptides synthesized in a rat hepatoma cell line. Quantitative two-dimensional gel analysis

To examine the degree of clonal heterogeneity in the synthesis of polypeptides in neoplastic cells, single-cell subclones from the rat hepatoma cell line H4-II-E were isolated. Polypeptides from the clones were resolved on high resolution two-dimensional polyacrylamide gels (PAGE), and quantitatively analyzed with a computerized two-dimensional PAGE analysis system developed in this laboratory. Only four qualitatively different spots were found which were synthesized in one of the subclones in four out of five experiments. In contrast, 5-20% of the spots showed statistically significant quantitative differences when any one subclone was compared to any other. These differences were generally quite small, averaging about 1.5-fold in intensity, although variations of fourfold or more were observed. Different cultures of the same subclone showed quantitative differences of the same order as seen in different subclones, indicating that this variability was primarily intraclonal in nature, i.e. associated with the cultures rather than the subclones. The distribution of quantitatively variable spots indicates that 50% or more of the polypeptides in these cells may display intraclonal variability. Similar results were obtained with a second set of subclones derived from these primary ones. Time course studies were conducted where cells were maintained continuously for 12 weeks, with samples taken for two-dimensional PAGE analysis once a week. The fraction of polypeptides that vary significantly generally increased with time between sampling points. Experiments with independent cultures grown in parallel indicate that about 4% of this variability can be correlated to the age of the culture media, although the majority appears due to uncontrolled and/or random differences that arise between cultures. These results indicate that independent cultures quickly develop detectable quantitative differences in the expression of a large fraction of their polypeptides. These differences cannot, at present, be associated with the observable biology of the cells and probably reflect time-associated variations in the balance of cellular macromolecular synthesis which arise in tissue culture cells.     

Loss of c-Met Disrupts Gene Expression Program Required for G2/M Progression during Liver Regeneration in Mice

Background

Previous work has established that HGF/c-Met signaling plays a pivotal role in regulating the onset of S phase following partial hepatectomy (PH). In this study, we used Met^fl/fl;Alb-Cre^+/− conditional knockout mice to determine the effects of c-Met dysfunction in hepatocytes on kinetics of liver regeneration.

Methodology/Principal Finding

The priming events appeared to be intact in Met^fl/fl;Alb-Cre^+/− livers. Up-regulation of stress response (MAFK, IKBZ, SOCS3) and early growth response (c-Myc, c-Jun, c-Fos, DUSP1 and 6) genes as assessed by RT-qPCR and/or microarray profiling was unchanged. This was consistent with an early induction of MAPK/Erk and STAT3. However, after a successful completion of the first round of DNA replication, c-Met deficient hepatocytes were blocked in early/mid G2 phase as shown by staining with phosphorylated form of histone H3. Furthermore, loss of c-Met in hepatocytes diminished the subsequent G1/S progression and delayed liver recovery after partial hepatectomy. Upstream signaling pathways involved in the blockage of G2/M transition included lack of persistent Erk1/2 activation and inability to up-regulate the levels of Cdk1, Plk1, Aurora A and B, and Mad2 along with a defective histone 3 phosphorylation and lack of chromatin condensation. Continuous supplementation with EGF in vitro increased proliferation of Met^fl/fl;Alb-Cre^+/− primary hepatocytes and partially restored expression levels of mitotic cell cycle regulators albeit to a lesser degree as compared to control cultures.

Conclusion/Significance

In conclusion, our results assign a novel non-redundant function for HGF/c-Met signaling in regulation of G2/M gene expression program via maintaining a persistent Erk1/2 activation throughout liver regeneration.     

Preparation of soybean seed samples for FT-IR microspectroscopy

Typical preparation of seed samples for infrared (IR) microspectroscopy involves imbibition of the seed for varying time periods followed by cryosectioning. Imbibition, however, may initiate germination even at 4° C with associated changes in the chemistry of the sample. We have found that it is possible to section seeds that are sufficiently hard, such as soybeans, on a standard laboratory microtome without imbibition. The use of dry sectioning of unimbibed seeds is reported here, as well as a comparison of different mounting media and modes of analysis. Glycerol, Tissue-Tek, and ethanol were used as mounting media, and the quality of the resulting spectra was assessed. Ethanol was the preferred mountant, because it dried quickly with no residue and thus did not interfere with the spectrum of interest. Analysis in transmission mode using barium fluoride windows to hold the samples was compared with transmission-reflection analysis with sections mounted on special infrared-reflecting slides. The two modes of analysis performed well in different regions of the spectrum. The mode of analysis (transmission vs. transmission-reflection) should be based on the components of greatest interest in the sample.